Heretofore known common methods for causing proteins to bind to carriers such as microplates, microbeads or sensor chips include hydrophobic bonding, covalent bonding, and the like. Hydrophobic bonding depends on the interaction between a hydrophobic surface of the carrier and a hydrophobic portion of the protein and it is convenient in that it requires no special reagent; on the other hand, it generally presents a weak binding force and if it is used in ELISA (enzyme-linked immunosorbent assay) or the like, washing and other operations that are performed after the binding often cause the protein to come off the carrier. It is also known that when proteins and carriers are bound together by hydrophobic bonding, most of the proteins lose their functions either completely or partially. In contrast, covalent bonding presents a strong binding force because it depends on the interaction between functional groups (e.g. amino groups) in the protein and functional groups (e.g. carboxyl groups) provided on the carrier's surface. However, when proteins and carriers are bound together by covalent bonding, most of the proteins have their functions lost either completely or partially as in the case of hydrophobic bonding.
In addition to hydrophobic bonding and covalent bonding, a method is known in which a plurality of histidines are fused to a terminal end of protein and the fused protein having such histidine tags is bound to a substrate such as a protein chip that has nickel provided on its surface. However, the interaction between the histidine tag and the nickel ion is not very strong and, what is more, the nickel ion which is known to bind nonspecifically to various biomolecules is not necessarily an all-purpose tool.
Avidin is a glycoprotein derived from egg white and it binds to biotin (vitamin H) extremely strongly. The interaction between avidin and biotin is one of the strongest modes of non-covalent bonding (Green (1975) Adv Protein Chem 29: 85-133). Streptavidin is an avidin-like protein derived from actinomycetes and it also strongly binds to biotin. So far, the (strept)avidin-biotin interaction, because of its strong force, has been extensively used in the fields of molecular biology and biochemistry for such purposes as the detection of antigens and antibodies (Green (1990) Methods Enzymol 184: 51-67).
Methods have been devised that bind proteins to carriers by making use of the above-described biotin binding ability of avidin or streptavidin. One example is a method in which (strept)avidin is fixed to a substrate such as a microplate by covalent or hydrophobic bonding and a biotinylated protein is then immobilized by binding to the (strept)avidin. In this method, however, the activity of avidin per se is lost partially and, what is more, the specific activity of the protein bound via biotin decreases, resulting in an action efficiency that is by no means satisfactory.
In contrast, a technique has been reported in which an avidin protein is first bound to a biotin-bound substrate by forming avidin-biotin bonds, to which a desired biotinylated protein is bound to ensure that avidin is bound to additional biotin pockets, whereby biotin, avidin, biotin, and the desired protein are fixed in that order to the substrate (JP 4-236353 A1). However, this method involves the step of biotinylating the desired protein, which results in the need for extra labor; another problem is the need to take the efficiency of biotin labeling into consideration.
Heretofore, with a view to using them as a protein labelling, a diagnosis marker, or a cell-specific targeting factor, fused proteins using avidin or streptavidin have been prepared (Airenne et al. (1999) Biomol Eng 16:87-92). In particular, the fused proteins prepared by fusing avidin or streptavidin to antibodies such as scFV or Fab fragments and IgG have been studied for their potential application in the specific targeting of drugs to cancer cells and the like. In addition, the idea has been described of a column that uses a streptavidin-scFv fused protein to fix scFv via avidin-biotin bonds (Kiprivanov et al. (1995) Hum Antib Hybrid 6: 93-101 and Dubel et al. (1995) J Immunol Methods 178: 201-209). However, avidin and streptavidin are difficult to express in E. coli as a soluble form in high yield; what is more, there has been no report in which an avidin-fused protein or a streptavidin-fused protein is immobilized by being bound to a biotinylated carrier so as to improve the activity of the proteins in comparison with the conventional binding methods. On the contrary, it has been reported that when a fusion protein of streptavidin and β-galactosidase was bound to biotinylated beads, the specific activity of β-galactosidase decreased to about 50% (Huang et al. (1996) Enzyme and Microbial technology.)
Patent Document 1: JP 4-236353 A1
Patent Document 2: WO02/072817
Patent Document 3: PCT/JP2006/326260
Patent Document 4: PCT/JP2006/304993
Non-patent Document 1: Green (1975) Adv Protein Chem 29: 85-133
Non-patent Document 2: Green (1990) Methods Enzymol 184: 51-67
Non-patent Document 3: Airenne et al. (1999) Biomol Eng 16: 87-92
Non-patent Document 4: Kiprivanov et al. (1995) Hum Antib Hybrid 6: 93-101
Non-patent Document 5: Dubel et al. (1995) J Immunol Methods 178: 201-209
Non-patent Document 6: Huang et al. (1996) Enzyme and Microbial technology
Non-patent Document 7: Hofmann et al. (1980) Proc Natl Acad Sci USA 77: 4666-4668
Non-patent Document 8: Iba et al. (1997) Gene 194: 35-46
Non-patent Document 9: Ideno et al. (2004) Appl Microbiol Biotechnol 64: 99-105
Non-patent Document 10: Kada et al. (1999) Biochim. Biophys. Acta., 1427: 33-43